| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
REPRODUCTION-DEVELOPMENT |
Department of Veterinary Biosciences (C.A.O., Q.Z., K.C., R.N., D.E.K., G.L.J., M.N., R.A.H.), University of Illinois, Urbana, Illinois 61802; and Departments of Morphology and Physiology (C.A.O., L.R.F.), Federal University of Minas Gerais, Belo Horizonte-MG, Brazil 31270-901
Address all correspondence and requests for reprints to: Dr. Rex A. Hess, Veterinary Biosciences, University of Illinois, 2001 South Lincoln, Urbana, Illinois 61802-6199. E-mail: . r-hess{at}uiuc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
knockout mouse. In agreement with this conclusion, there was a decrease in Na+-H+ exchanger-3 mRNA and protein, which is consistent with previous studies showing that ER is required for expression of Na+-H+ exchanger-3 and ultimately fluid reabsorption in the efferent ductules. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ERKO) or both ER and ERß (ßERKO) has demonstrated that ER is essential for fertility in the male and that estrogen has a role in the regulation of the male reproductive tract (1, 2, 3, 4, 5). In the ERKO and antiestrogen-treated mice, efferent ductules, which are small tubules that transport sperm from the rete testis to the epididymis, showed the most severe histopathological changes following ER disruption (1, 6, 7, 8). This is not surprising because this region of the male reproductive system expresses the highest concentration of ER-, which is even higher than that found in normal female tissues (9, 10, 11, 12, 13, 14). The lumen of efferent ductules in ERKO and antiestrogen-treated mice were dilated and the epithelium was regressed. There was clear evidence that disruption of ER in the male mice results in the inhibition of fluid reabsorption in efferent ductules (1, 6, 7, 8).
Efferent ductules are responsible for the reabsorption of more than 90% of the fluid leaving the testis (15). This important physiological function is similar to that in the proximal tubules of the kidney, involving the active transport of Na+ from the lumen toward the basement membrane and passive movement of water toward the ion concentration gradient (16). This mechanism includes the participation of a Na+,K+-ATPase along the basolateral membrane (17), the Na+-H+ exchanger-3 (NHE3) and aquaporin-1 on the microvillus border (18, 19, 20, 21, 22) and carbonic anhydrase (CAII) in the apical cytoplasm (23, 24). In a recent study, NHE3 was shown to be the major protein regulated by estrogen to mediate fluid reabsorption in the efferent ductules (25). Effects of ER disruption on aquaporin-1 and CAII appeared to be secondary because their mRNAs were not decreased, but that of NHE3 was reduced by 60–80% in ERKO and ICI 182,780-treated mice.
Although ICI 182,780 treatment in the adult mouse results in male reproductive tract effects similar to the ERKO mouse (7), the ERKO male does contain developmental defects that persist in the adult tissues (8). Furthermore, differences between species in the distribution of ER and ERß in the male reproductive tract have been reported (8, 10, 11, 26), and sensitivity to estrogens and antiestrogens has been found to differ among species and animal strains (27, 28, 29, 30, 31, 32). Therefore, further development of the ICI-treatment model in the adult male remains an important goal and the rat was selected as an alternative species (33). The antiestrogen ICI 182,780, which is a steroidal antiestrogen that binds to both ER and ERß (34, 35) and does not cross the blood-brain barrier (36), was used for an extended period of treatment (100 and 150 d) in the rat model. This long-term treatment reproduced the male reproductive tract phenotype seen in ERKO, including testicular atrophy and infertility (33). The success of ICI 182,780 treatment in adult male rats substantiated the role of estrogen in the male tract without the problems associated with developmental absence of ER, which is found in the knockout mice. However, it was still unclear whether the changes in testis and efferent ductules resulted from primary or secondary effects of treatment. These results prompted us to evaluate the time-response effects of ICI 182,780 to determine the sequence of events leading to infertility and testicular atrophy and to test the hypothesis that estrogen regulates the NHE3 in efferent ductules of rat. The focus of this study was on testicular and efferent ductule effects because prior studies have shown these regions to be major sites of pathological change following the disruption of ER in the male.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Treatment
Beginning at 30 d of age, the rats were treated once a week with sc injections of a long-acting formulation of ICI 182,780 (Faslodex, kindly provided by AstraZeneca, Macclesfield, UK), at a dosage of 10 mg/animal in a volume of 0.2 ml vehicle. This dosage was found to be effective for inducing effects on rat efferent ductule without inducing significant differences on body and sexual gland weights (33). The control group received the same volume of castor oil. Matched control and ICI-treated rats (three animals per group) were euthanized on d 3, 15, 45, 52, 73, 100, and 150 after the first injection for the histological study; on d 7, 15, 45, and 100 for immunohistochemistry (three animals per group); on d 45 for acid phosphatase detection (two animals per group); and on d 3 and 55 for Northern blot analysis (25 animals per group, with pooled tissues in each group). Because rats are still growing at 30 d of age and some developmental effects of ICI 182,780 could occur, we treated a separate group of four mature adult male rats (90 d old) for comparison of the effects of ICI 182,780 treatment beginning at full maturity (treated for 45 d) with those at 30 d old.
Histology
The rats were anesthetized (ip sodium pentobarbital 0.1 ml/100 g body weight), weighed, and perfused intracardially with 4% (wt/vol) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The testes, reproductive tract, ventral prostate, and seminal vesicles (with coagulating glands) were removed, immersed in the same fixative, and stored at 4 C for further processing. After fixation, the testes were dissected from the efferent ductules, weighed, and cross-sectioned at the midrete testis. Weights of the accessory sex glands were also recorded after fixation. The efferent ductules as well as the testis sections were embedded in glycol methacrylate resin, sectioned at 2.5 µm, and stained with 1% toluidine blue or periodic acid-Schiff (PAS) with hematoxylin counterstain.
Morphometry
Histological sections were analyzed morphometrically using NIH image software (http://rsb.info.nih.gov/nih-image). Images were obtained using a Spot-2 digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) and plan apochromatic lenses (Olympus Corp. America Inc., Melville, NY) and processed using a MAC-G3 computer (Apple Computer, Cupertino, CA) with Adobe Photoshop (San Jose, CA).
The seminiferous tubule diameter and luminal area were measured in 10 randomly selected round cross-sections of tubules, at stages VII-VIII, except for testis on d 3 and 150. On d 3, spermatogenesis was still incomplete and the lumen was not patent in some seminiferous tubules. On d 150 most of the seminiferous tubules were atrophic.
The efferent ductule luminal diameter was determined by measuring the widest diameter of five sections of tubules from the proximal area. The proximal area was selected because past studies had shown a similar reaction in proximal and distal regions (our unpublished data), and the proximal region is responsible for reabsorption of approximately 70% of the fluid entering the efferent ductules (15). The epithelial height was measured in straight sections of the epithelium, in 25 cells/animal. Height of the microvillus border was measured in the same cells.
To estimate percent area of PAS-positive lysosome-like granules in nonciliated cells, images (Adobe Photoshop) were changed to CMYK and then adjusted using the channel mixer command (monochrome, cyan -200%, magenta +200%). The pictures were then opened with NIH image software, and an epithelial area composed of five consecutive nonciliated cells was outlined and measured. To discriminate the lysosomes from surrounding background, the density-slicing mode was used. Then the area occupied by the highlighted lysosomes was measured. The amount of lysosomal granules per 100 µm2 was calculated.
Immunohistochemistry
NHE3 was localized by immunohistochemistry in efferent ductules of treated and control animals on d 7, 15, 45, and 100. Tissue sections from treated and control animals at each time point were run in parallel, and all staining was replicated to confirm the results. Tissues were fixed by perfusion with neutral buffer formalin, embedded in paraffin, and stained using standard methods for microwave antigen retrieval. Sections were incubated for 12 h at 4 C with diluted (1:500) primary antibody, a polyclonal rabbit antirat NHE3 (Chemicon International, Temecula, CA). For negative controls, the sections received PBS in place of the primary antibody. Renal tissue was used for positive control. After washing in PBS, sections were incubated with a secondary antibody, biotinylated goat antirabbit IgG, diluted 1:100 (DAKO Corp., Glostrup, Denmark) for 1 h at room temperature, and then incubated with the avidin-biotin complex (Vector Laboratories, Inc., Burlingame, CA) for 30 min. Reactivity was visualized using DAB chromogen. Sections were counterstained with Mayer’s hematoxylin.
Northern blot analysis
Tissues for total RNA analysis on d 3 and d 55 after the first injection were collected and pooled from 25 control and 25 treated rats, respectively. Total RNA was isolated from efferent ductules by the guanidinium isothiocyanate/phenol chloroform method. After electrophoresis of total RNA (20 µg) on a 1.5% formaldehyde denaturing agarose gel and blotting to Duralon-UV membranes (Stratagene, La Jolla, CA), the membrane was prehybridized in QuikHyb solution (Stratagene) for 2 h and then hybridized with denatured 32P-labeled probes and sonicated salmon sperm DNA at 58 C overnight. After hybridization, the filters were washed twice for 5 min each at room temperature in 2x sodium citrate-buffered saline-0.1% SDS, followed by a 10-min wash in 0.1x sodium citrate-buffered saline-0.1% SDS at 65 C. The NHE3 probe was a PCR product from rat NHE3 cDNA plasmid. Primers for NHE3 were 5'-TGGATTTCCTGCTATTTGGC-3' and 5'-TCGCTCCTCTTCACCTTCA-3' (GenBank accession no. M85300, nt615-1485). The Northern blots were normalized for variations in loading by a final hybridization with the 36B4 probe, which is not influenced by estrogen treatment (38). Relative quantification was made by digitizing images using UN-SCAN-IT (Silk Scientific, Orem, UT).
Hormone measurement
Plasma levels of testosterone and LH were estimated by RIA. Blood samples were obtained by cardiac puncture immediately before death. The plasma was separated by centrifugation and stored at -20 C for subsequent hormone assays. All samples were measured in duplicate.
Plasma concentration of LH was estimated by a double antibody method, using rat LH-RIA immunoreagents (antirat LH-S-11 antisera, rat LH-I-9 IOD, and rat LH-RP-3) and procedures provided by the National Hormone and Pituitary Program (NIDDK, Rockville, MD). The NIDDK rat LH-RP-3 preparations were used as standards. The assay sensitivity was 0.2 ng/ml and the intraassay coefficient of variation was 8%.
Plasma testosterone level was measured using an antibody to 4- androsten-11, 17ß-diol-3-one-11-hemisuccinate. BSA (antitestosterone 1969–11B) developed by Dr. O. D. Sherwood (University of Illinois, Urbana, IL) (39). Cross-reactivity with E2 and estrone was less than 0.0001 (0.01%) each. The only significant cross-reactivity of the antisera was with 5-androstan-17ß-ol-3-one (5-DHT) (52%). However, because of the low circulating levels of 5-DHT in the male rat (40, 41), the interference of DHT is considered to be negligible (42); therefore, the results were expressed as nanograms of testosterone. For the assay, plasma was extracted with toluene:petroleum ether (2:5 vol/vol). The efficiency of the extraction was 86.5%. The limit of detection was 0.2 ng/ml and the intra- and interassay coefficients of variation averaged 6.6% and 2.8%, respectively. The reported plasma concentrations of testosterone were corrected for recovery.
Acid phosphatase activity
For identification of lysosomes, the Gomori histochemical method for acid phosphatase was used (43). Efferent ductules from d 45 ICI-treated and control rats were fixed by perfusion with neutral buffer formalin, frozen in liquid nitrogen and stored at -20 C. Liver tissue was used as positive control. Frozen sections (8 µm) were incubated for 20 min at room temperature in the medium with Na-ß-glycerophosphate (Sigma, St. Louis, MO) as a substrate. Following incubation, the sections were washed in distilled water and transferred to 1% ammonium sulfide solution to develop the stain. The reaction was monitored microscopically and was stopped by immersion in distilled water when a dark brown positive reaction was observed. As negative control, slides were incubated in the medium without the Na-ß-glycerophosphate substrate. To assure that the observed reaction was specific, an inhibition test was performed by adding to the incubation medium sodium fluoride (10 mM), which is a specific inhibitor of the acid phosphatase activity.
Statistics
Data were analyzed by two-way ANOVA to compare the differences between treated and control groups. Multiple post hoc comparisons was performed by a Newman-Keuls test to detect differences at individual time points (P < 0.05). Results are presented as mean ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). There were no treatment differences in body weight (Table 1). Weight of the ventral prostate and seminal vesicle-coagulating glands also showed no difference between controls and ICI-treated males (Table 1), neither in absolute weights of the glands nor weight relative to body weight.
|
and 2). The overall increase in luminal diameter of the efferent ductules proximal region was 199.4%. Luminal dilation was first observed at d 3 post treatment in some ductules. Thereafter the ductules had statistically significant and progressive dilation, showing maximum dilation on d 73 and remaining dilated until d 150. Coincident with the efferent ductule dilation, a significant decrease in the epithelial height occurred following the ICI 182,780 treatment (Fig. 1B). Starting from d 15, epithelial height was significantly less than controls. A gradual decrease in epithelial height over time was observed in the ICI-treated ductules, with the mean value reaching a plateau on d 52. Although a similar mean height was found in treated animals from d 52 to d 150, there was a large variation in height in some epithelial areas, even in the same tissue section (Fig. 3). Compared with the controls, the maximum decrease in height (48% decrease) was found on d 150, when a large proportion of cuboidal cells were observed (Fig. 3). Conversely, a gradual increase in epithelial height was observed over time in control efferent ductules (Fig. 1B).
|
|
|
). However, there was a clear trend for increased microvillus border height from the beginning of the ICI 182,780 treatment until d 73, whereas thereafter a decrease in microvillus border height was detected, culminating with a 39% decrease at d 150. The only exception was seen on d 52, when a lower microvillus border was found for both treated and control animals, suggesting a potential problem with tissue fixation. Coexistence of areas with variable microvillus border height was frequently seen in the treated efferent ductule epithelium from d 73 to d 150 (Fig. 3).
Beginning on d 15 post treatment and continuing until d 100, there was a significant increase in the percent area occupied by PAS-positive granules in 100 µm2 of the nonciliated cell cytoplasm, compared with controls (Figs. 1D and 3). The granules were identified as part of the lysosomal compartment, based on their acid phosphatase activity. The maximum effect on lysosomes occurred on d 52 when there was a 172% increase in lysosomal area per 100 µm2 area of cytoplasm. After d 73, the lysosomal area in ICI-treated tissues decreased, and on d 150 was lower than controls (64% decrease). In the treated group, lysosomes were redistributed within the cytoplasm (Fig. 3). They were surrounding the nucleus and occupying the basal cytoplasm, whereas in control nonciliated cells, lysosomes were fewer than treated group and seen in the supranuclear and apical cytoplasm. The distal efferent ductules contained fewer lysosomes in both control and treated animals. Early endocytotic components of the lysosomal system, which were identified by toluidine-blue and PAS staining as clear vesicles in the adluminal cytoplasm, were evident in control animals as well as in ICI-treated cells. However, these vesicles appeared decreased in some cells after d 73 of treatment and almost disappeared at d 150.
Rete testis
At all time periods after d 3, the rete testis lumen was dilated following ICI 182,780 treatment, compared with controls (Fig. 4). At d 3 the rete testis was dilated in only one of three animals. Rete testis dilation increased over time with ICI 182,780 treatment. The rete testis epithelium did not show consistent changes in cell height or morphology after treatment.
|
). However, on d 45, the weight of testes in ICI-treated males increased rapidly over controls and continued heavier until d 100. The increase in testis weight on d 45 of ICI 182,780 treatment was accompanied by dilation of the seminiferous tubule lumen, which was evident from d 52 to d 100, independent of the cycle stage of the seminiferous epithelium (Figs. 5 and 6). After d 100, a sharp decrease in testis weight was seen, compared with controls. No remarkable histological difference was noted in the seminiferous epithelium between controls and ICI-treated rats, until d 150 when most tubules were atrophic in the treated rats (Fig. 6).
|
|
). There was a gradient of NHE3 staining, increasing from proximal to distal regions of the ductules. Immunoexpression of NHE3 was decreased slightly on d 7 in nonciliated cells of the treated efferent ductules. The staining intensity continued to decrease over time until staining was barely detectable on d 100 of ICI 182,780 treatment (Fig. 7). In agreement with the immunohistochemical results, NHE3 mRNA was reduced 98.5% in ICI-treated animals, compared with controls on d 55 post treatment (Fig. 8) but on d 3 the message was not reduced (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ERKO mouse (7, 8); however, the developmental sequence of changes in the ERKO male tract has not been reported.
Long-term effects of ICI 182,780 resembled an ERKO-like reproductive tract phenotype and culminated in testicular atrophy and infertility in the rat (33). Here we describe for the first time the sequence of changes in the efferent ductules and testis that preceded the terminal events of testicular atrophy. Additionally, it is shown that the mechanism by which estrogen regulates fluid reabsorption is related to the expression of NHE3 in nonciliated cells of the efferent ductules. It is noteworthy that all changes seen in efferent ductules after antiestrogen treatment occurred without effects on the concentration of plasma testosterone. This observation reinforces the importance of estrogen in the male reproductive tract, especially within this unique region whose function appears to be essential for fertility.
Testosterone and LH concentrations in blood plasma were not changed following ICI 182,780 treatment. Body and accessory sex gland weights were also unaffected at all time points, demonstrating that ICI 182,780 had a specific peripheral action in the male rat reproductive tract. Plasma E2 concentrations were not determined because of difficulties in separating ICI 182,780 from endogenous blood estrogens. However, because the parameters above are susceptible to serum levels of E2 (44) and they were similar to controls, it is unlikely that E2 levels were altered. In contrast to ICI-treated rats, serum LH (45) and testosterone levels were increased in ERKO mice (3, 45). Aromatization of testosterone to E2 and a functional ER has been shown to be essential in the negative feedback action of androgens in the male hypothalamus-pituitary axis (26, 45, 46, 47, 48). Therefore, the difference in hormone levels between the ERKO mouse and ICI-rat can be explained at least in part because ER- mediated action is absent in the ERKO brain but not in the ICI-treated rat because ICI 182,780 does not cross the brain barrier (36).
There was an unprecedented increase of 200% in luminal diameter in the efferent ductules after ICI 182,780 treatment. Luminal dilation represented one of the first effects of ICI 182,780 treatment as seen on d 3. In ERKO mice, dilation of efferent ductules was also a primary effect of ER disruption (7, 8); however, dilation of the efferent ductules was greater in ICI-treated mice of the same age (7). Surprisingly, aromatase knockout mice (49) and aromatase inhibitor-treated rats (50) did not show such response, indicating that a functional ER is required for maintenance of efferent ductule fluid physiology. The apparent added increase in luminal diameter following antiestrogen treatment is probably owing to effects of ICI 182,780 on Cl- secretion because the ligated ERKO ductule in vitro showed 40% increase in diameter (1). Recent studies have also shown an increase in mRNA for the chloride channel, cystic fibrosis transmembrane regulator (CFTR), in ERKO and ICI-treated male rat efferent ductules (51).
Conversely to the dilation of efferent ductules, the rete testis dilation was greater in the ERKO mouse than after ICI 182,780 treatment in mouse or rat (7, 33). This difference is most likely owing to developmental abnormalities in the ERKO (7). In the rat, the rete testis was not dilated in two of three animals on d 3, which is indicative that the dilation of efferent ductules preceded the dilation of the rete testis. In addition, the efferent ductule dilation was present before seminiferous tubule dilation and persisted until the end of the experiment, when the testes became atrophic. These results are consistent with the hypothesis that dilation of the efferent ductules occurs because of a decrease in efferent ductule fluid reabsorption, which leads ultimately to the accumulation of fluid in the testis (1, 52).
In the present study, a transient increase in testicular weight occurred from d 45 to d 100. This response was similar to that described for the ERKO mouse from d 32–45 to d 90–100 (1). In the rat, the increase in testis weight correlated in time with dilation of seminiferous tubules from d 52 to d 100. It was during this time period that the efferent ductules reached their maximum dilation. Therefore, it is reasonable to postulate that the maximum accommodation of fluid in the efferent ductules was achieved before the buildup of fluid within the testis, which would induce subsequent swelling of the testis, followed by atrophy of the seminiferous epithelium (1, 33, 52). Similar to the effects seen in the testes of ICI-treated rats, which took 150 d to become atrophic (this study and 33), testicular disruption required an extended period of time to be manifested in the ERKO and ßERKO males (3, 5). Disruption of spermatogenesis was also delayed in the aromatase knockout males (49).
The efferent ductules displayed severe morphological effects in the ERKO and ICI-treated mice (1, 7, 8). However, in the ICI-treated rat, the epithelial cells showed less reduction in height than was seen in the mice and the height stabilized after 52 d of treatment, which coincided with the time period during which the efferent ductule lumen reached its maximum dilation. The ERKO-like effect on the epithelium was not observed in the rat until the end of the experiment on d 150. However, the degree of luminal dilation does not appear to cause the epithelial disruption because it was recently shown that the efferent ductule lumen was dilated even to a greater extent in the NHE3 knockout mouse, but its ductal epithelium was columnar and normal in every respect (25). Therefore, a difference between these two rodent species in response to antiestrogen treatment is evident.
The microvillus border of nonciliated cells of the efferent ductules was decreased in height or often missing in ERKO and ICI-treated mice (1, 6, 7, 8). However, in the present study, the microvillus border showed large variations in height, with an increase in the height up to d 73 but an overall decrease thereafter. A taller microvillus border is suggestive that the epithelium is attempting to compensate for the inhibition of fluid reabsorption. Such a response would be similar to the NHE3 knockout mouse and CAII-deficient mice, which had efferent ductule with greater dilation and taller epithelium than ERKO (25). We have no explanation for the differences between mice and rats in response to ICI 182,780, but it is clearly evident that disruption of ion and water transport, which accounts for the fluid accumulation in the efferent ductules (25), is independent of the effects on epithelial morphology.
Evidence that an Na+/H+ exchanger is the principal physiological mechanism responsible for fluid and electrolyte reabsorption in the rat efferent ductules has already been shown by microperfusion in vivo (19). In that study, approximately 70% of fluid reabsorption was dependent on this exchanger. Confirming the physiology, an active NHE3 protein was detected in the apical membrane of rat efferent ductules nonciliated cells (20, 21). In the ICI-treated rat efferent ductules, NHE3 protein was beginning to show decreased expression by d 7 and the reduction became greater over time, reaching levels nearly undetectable by d 100. This decrease was consistent with Northern blot analysis showing a decrease of 98.5% in the NHE3 mRNA on d 55. Although we did not determine the exact date for effects on NHE3 mRNA, it is likely between d 3 and d 7 when the protein began to decline. As in the mouse, in which NHE3 expression and Na+ transport have been shown to be regulated by estrogen (25), loss of this important ion exchanger was independent of apical cytoplasmic disruption. It is unlikely that the decrease of NHE3 protein (as early as d 7 after treatment) and mRNA (55 d post treatment) is owing to the loss of the apical microvillus brush border because microvillus height is greater than controls up to d 73.
Although the increase in luminal diameter was not significant on d 3, it is interesting that some lumens began dilating so rapidly before an overall change in NHE3 expression. Possible explanations include an increase in secretion by testis or the efferent ductule epithelium. However, on d 3 the animals were only 33 d old and the testicular secretions had not reached normal levels, as evidenced by the lack of opening of the seminiferous tubule lumens in many tubules. Thus, the efferent ductules were not at this time under normal estrogen and androgen stimulation from the lumen. Also, our previous study showed that in the ERKO male, there was no increased testicular secretion, but rather a decrease was observed (1). The same study also found that in vitro ligated efferent ductule segments increased in luminal area, suggesting that in addition to regulating fluid reabsorption, estrogen in the male tract also regulated secretion, possibly of Cl- through CFTR, which is expressed in the efferent ductule epithelium (53). Another explanation is found in the potential for ICI 182,780 to inhibit both ER and ERß in this tissue (54). A recent study found that ICI treatment of mice increases CFTR mRNA (51). Treatment of both the rat and the mouse with ICI 182,780 resulted in proportionally greater dilation of the efferent ductule lumen than in the ERKO mouse (1, 7, 33). Therefore, it is reasonable to propose that this pure antiestrogen is inhibiting NHE3 as well as other factors responsible for ion and fluid reabsorption. Thus, the first response to ICI 182,780 may be increased Cl- secretion, which would cause slight dilation of the ductule lumen, followed by decreased expression of NHE3 with subsequent loss of Na+ and water transport, ultimately resulting in even greater dilation of the lumen.
The exchange of Na+ and H+ at the apical surface does not account for 100% of fluid reabsorption in the rat efferent ductules (19). Thus, other mechanisms, such as the Na+,K+-ATPase along the basolateral membrane (17), could be inhibited by the ICI 182,780 treatment. However, previous studies by our laboratory have shown that the general activity of Na+,K+-ATPase was dependent on circulating androgens and not estrogen (16). Furthermore, others have found that ICI treatment increases the expression of the Na+/K+,ATPase-1 mRNA (51). Nevertheless, reabsorption of fluid in the efferent ductules is rapid (55) and essential for fertility (33); therefore, the existence of multiple mechanisms that could fine-tune this physiological process would be expected. The role that an overall balance of estrogen and androgens would play in this system remains to be determined.
The transient increase in lysosomal granules and height of microvilli in rat nonciliated cells of the efferent ductules were major differences from the ERKO and ICI-treated mice. Microvilli and the endocytotic apparatus, including lysosomes, were reduced to insignificant levels in the ERKO and ICI-treated mice (1, 6, 7, 8). In the rat, percent area of the lysosomes reached a peak between d 52 and d 73, but then there was a dramatic reduction by d 150. The cause of this difference in response between mice and rats is not known. The decrease in lysosomes in the rat corresponded to the induction of testicular atrophy; therefore, it is possible that the maintenance of lysosomes by the rat epithelium is dependent on active spermatogenesis and a corresponding luminal fluid milieu that is associated with the passage of sperm through the efferent ductules.
The rodent data clearly show that estrogen is important in the male reproductive tract. However, clear evidence of estrogen function in man has not been reported. An adult man with homozygous null mutation for ER has been identified, but semen analysis was not reported (56). On the other hand, two aromatase-deficient men have been described both showing some effect on reproductive phenotype. Although direct extrapolation of data from the rodent to human is not possible, it is intriguing to consider that the youngest aromatase-deficient man (24 yr of age) had increased testis size (57), but the oldest (38 yr) showed reduced testicular size, severe azoospermia, and infertility (58, 59). These findings suggest that estrogens are also important for reproduction in man. The observations seen in the present study and in ERKO males (1) recapitulate mechanisms that could account for an increase in testis size in the younger aromatase-deficient man, but decreased size in the older man having a deficiency in estrogen synthesis.
In conclusion, this study provides further evidence that estrogen is involved in the regulation of structure and function of the efferent ductules and that over time a functional ER is required for the expression of NHE3 as well as for maintenance of ion and fluid reabsorption, an essential function for long-term fertility in the rodent.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: 5-DHT, 5-Androstan-17ß-ol-3-one; ERKO, knockout mouse ER-; CAII, carbonic anhydrase; CFTR, cystic fibrosis transmembrane regulator; NHE3, Na+-H+ exchanger-3; PAS, periodic acid-Schiff.
Received October 16, 2001.
Accepted for publication February 26, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER ) and ß (ER ß) on mouse reproductive phenotypes. Development 127:4277–4291[Abstract]
knockout male mouse. Anat Rec 263:10–18[CrossRef][Medline]
has a functional role in the mouse rete testis and efferent ductules. Biol Reprod 63:1873–1880 knockout mice. J Androl 21:107–121[Abstract]
and ß) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 18:602–611 within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood. J Endocrinol 153:485–495 and ß proteins in the testes and male reproductive system of human and non-human primates. Mol Hum Reprod 7:227–236 messenger RNA in male reproductive organs of adult goats. Biol Reprod 64:1432–1438 in sex ducts and gonads of newborn piglets. Histochem Cell Biol 115:521–526[Medline]
knockout mice. Endocrinology 139:4092–4101 and ERß. Biol Reprod 65:1534–1541 and ß. Endocrinology 138:863–870This article has been cited by other articles:
 |
|
 |
|
  C. A. Oliveira, A. B. Victor-Costa, and R. A. Hess Cellular and Regional Distributions of Ubiquitin-Proteasome and Endocytotic Pathway Components in the Epithelium of Rat Efferent Ductules and Initial Segment of the Epididymis J Androl, September 1, 2009; 30(5): 590 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Yasuhara, G. R. O. Gomes, E. R. Siu, C. I. Suenaga, E. Marostica, C. S. Porto, and M. F. M. Lazari Effects of the Antiestrogen Fulvestrant (ICI 182,780) on Gene Expression of the Rat Efferent Ductules Biol Reprod, September 1, 2008; 79(3): 432 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M L Gould, P R Hurst, and H D Nicholson The effects of oestrogen receptors {alpha} and {beta} on testicular cell number and steroidogenesis in mice Reproduction, August 1, 2007; 134(2): 271 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J Meachem, S. Schlatt, S. M Ruwanpura, and P. G Stanton The effect of testosterone, dihydrotestosterone and oestradiol on the re-initiation of spermatogenesis in the adult photoinhibited Djungarian hamster J. Endocrinol., March 1, 2007; 192(3): 553 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Stabile, M. Russo, and P. Chieffi 17{beta}-Estradiol induces Akt-1 through estrogen receptor-{beta} in the frog (Rana esculenta) male germ cells. Reproduction, September 1, 2006; 132(3): 477 - 484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Da Silva, C. Silberstein, V. Beaulieu, C. Pietrement, A. N. Van Hoek, D. Brown, and S. Breton Postnatal Expression of Aquaporins in Epithelial Cells of the Rat Epididymis Biol Reprod, February 1, 2006; 74(2): 427 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J Meachem, D. M Robertson, N. G Wreford, R. I McLachlan, and P. G Stanton Oestrogen does not affect the restoration of spermatogenesis in the gonadotrophin-releasing hormone-immunised adult rat J. Endocrinol., June 1, 2005; 185(3): 529 - 538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Pais, I. Leav, K.-M. Lau, Z. Jiang, and S.-M. Ho Estrogen Receptor-{beta} Expression in Human Testicular Germ Cell Tumors Clin. Cancer Res., October 1, 2003; 9(12): 4475 - 4482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhou, R. Nie, G. S. Prins, P. T. K. Saunders, B. S. Katzenellenbogen, and R. A. Hess Localization of Androgen and Estrogen Receptors in Adult Male Mouse Reproductive Tract J Androl, November 1, 2002; 23(6): 870 - 881. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
0.05) at all time points post treatment. The maximum dilation occurred after 73 d of treatment. B, The height of the efferent ductules epithelium decreased after treatment, reaching a plateau on d 52. Conversely, the epithelium in control efferent ductules increased over time. C, Microvillus border of efferent ductule epithelia was taller in ICI-treated rats up to d 73; however, on d 100 and 150 a significant (*, P 
